The signal transduction mechanisms mediating responses to environmental stresses and inflammatory agents are poorly understood. Yet, these stimuli are responsible for a number of pathological conditions of clinical importance including acute phase septic shock, ischemic injury (as occurs in heart attack and stroke), fever and radiation damage. Recent results from the applicant's laboratory contributed to the delineation of a mammalian signaling pathway that mediates responses to environmental stresses, ischemic injury and the inflammatory cytokines TNF- alpha and IL-1. This signaling pathway uses three sequentially acting protein kinases. This module of enzymes is analogous to the modules that are a part of the mammalian mitogenic response pathway and several yeast signal pathways. The three enzymes of the stress activated module are MEK kinase 1 (MEKK1), SAPK/ERK kinase 1 (SEK1) and stress activated protein kinase (SAPK; an ERK family member). The transcription factor, c-Jun is a target of this pathway and SAPKs are the primary mediators of its phosphorylation in response to stress. The four aims of the proposed research will dissect the regulation and function of the SAPK pathway. Aim 1 is to identify molecular species that interact with the MEKK1 N-terminal regulatory domain and characterize their effects on MEKK1 activation of SEK1. These MEKK1 binding species will be identified by application of yeast two hybrid and affinity matrix approaches. Aim 2 employs a yeast two hybrid screen and biochemical assays to identify additional SAPK substrates. Aim 3 employs low stringency screening methods and stable and transient transfection to isolate other members of the MEKK family. Various analyses will be applied to determine if any of the novel MEKK members activate the SAPK pathway. Finally (Aim 4), the potential role of mammalian Ste20 homologs in the activation of the SAPK kinase module will be tested.